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Recombinant light chains of botulinum neurotoxins and light chain fusion proteins for use in research and clinical therapy

Inactive Publication Date: 2013-12-26
SMITH LEONARD A +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to the design and construction of synthetic DNA molecules that encode the seven light chains of Clostridium botulinum neurotoxin and are capable of being expressed in heterologous prokaryotic or eukaryotic hosts. The invention is based, in part, on modifying the wild-type BoNT sequence according to the codon usage normally found in genes that are highly expressed in the host organism. By selecting codons rich in G+C content, the synthetic DNA molecules may further be designed to lower the high A+T rich base composition found in clostridial genes. The invention also provides methods of expressing and purifying recombinant BoNT light chains, and methods and compositions for eliciting an immune response to BoNT LC and BoNT HN.

Problems solved by technology

The toxins primarily act by inhibiting the neurotransmitter acetylcholine at the neuromuscular junction, causing paralysis.
The most common immediate cause of death is respiratory failure due to diaphragmatic paralysis.
Extreme toxicity of these toxins and their lability in purified preparations have limited any detailed characterizations.
Their proteolysis inhibits exocytosis and blocks acetylcholine secretion, leading ultimately to muscular paralysis.
However, in digitonin-permeabilized chromaffin cells, the LC inhibits exocytosis (Bittner et al., 1989), and direct microinjection of the LC into the cytosol results in blockage of membrane exocytosis (Bittner et al., 1989; Bi et al., 1995).
In spite of such immense importance, studies of the LC have been limited by its availability.
These preparations therefore retain some contaminating toxicity of the dichain, have low solubility, and often begin to proteolytically degrade and start losing activity within hours of storage in solution.
The LC of serotype A has been separated and purified from the full-length toxin by QAE-Sephadex chromatography from 2 M urea; however, the preparation suffers from low solubility (Shone and Tranter, 1995).
These preparations almost invariably contain contaminating full-length toxins, and the commercially available preparations precipitate from solution or undergo proteolytic degradation upon hours of storage in solution.
However, the poor expression of the cloned products, probably due to rare codon usage in clostridial DNA (Makoff et al., 1989, Winkler and Wood, 1988), remained a major hurdle in obtaining adequate amount of the protein for structural and functional studies.
Inactivation of SNAP-25, VAMP, or syntaxin by BoNT leads to an inability of the nerve cells to release acetylcholine resulting in neuromuscular paralysis and possible death, if the condition remains untreated.
However, recombinant methods as described in the publications above do not yield optimal results because botulinum codons are not translated well in other organisms commonly used for production, such as E. coli or yeast.
Furthermore, no easily translatable, recombinant form of the non-neurotoxic, mutated light chain presently exists.
These preparations, therefore, retain some contaminating toxicity, have low solubility, and undergo proteolytic degradation within hours and days of storage in solution.
The poor expression of the native gene was probably due to the high A+T composition found in the clostridial DNA.

Method used

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  • Recombinant light chains of botulinum neurotoxins and light chain fusion proteins for use in research and clinical therapy
  • Recombinant light chains of botulinum neurotoxins and light chain fusion proteins for use in research and clinical therapy
  • Recombinant light chains of botulinum neurotoxins and light chain fusion proteins for use in research and clinical therapy

Examples

Experimental program
Comparison scheme
Effect test

example 1

Chemicals, Buffers; and Reagents

[0068]Buffer T (20 mM Tris-HCl, pH 9.2) and buffer G (50 mM sodium glycine, pH 9.0) were used as indicated. SKL (sodium N-lauryl sarcosine or sarkosyl) was from Sigma. Highly purified (>95%) full-length BoNT / A was purchased from List Biologicals (Campbell, Calif.). Rabbit polyclonal antibodies against a 16-residue N-terminal sequence (PFVNKQFNYKDPVNGV; SEQ ID NO:1) of the BONT / A LC were produced and affinity purified by Research Genetics (Huntsville, Ala.). Peroxidase-coupled goat anti-rabbit and anti-mouse IgG (H+L) and ABTS substrate were from Kirkegaard Perry Laboratories (Gaithersburg, Md.). Oligonucleotides, designed for E. coli codon usage (Anderson and Kurland, 1990) and ranging in size from 70 to 100 nucleotides, were synthesized by Macromolecular Resources (Fort Collins, Colo.).

example 2

Construction and Expression of a Synthetic DNA Encoding rBoNT / A LC

[0069]The DNA encoding the enzymatic LC domain of BoNT / A was assembled from three segments, a 335-base pair (bp) Sal I-Sph I fragment, a 600-bp Sph I-Kpn I fragment, and a 460-bp Kpn I EcoR I fragment. To construct the first segment, six oligonucleotide pairs were annealed, ligated, and, after PCR amplification, inserted into pGEM3Zf at Sal I-Sph I restriction enzyme sites. The second segment was built by annealing and ligating eight oligonucleotide pairs, followed by its amplification and insertion into the Sph I and Kpn I sites of pGEM3Zf. The final segment was constructed by annealing and ligating six oligonucleotide pairs, followed by its amplification and insertion into the Kpn I-EcoR I sites of pGEM3Zf. Nucleotide sequencing of gene fragments in pGEM3Zf was performed to identify clones in each group with minimal misincorporations. In vitro mutagenesis was performed to correct the misincorporations in the BoNT / A ...

example 3

Fermentation

[0074]A frozen stock seed culture of recombinant E. coli harboring the synthetic DNA encoding the LC of BoNT / A was grown at 37° C. to an OD600 of 2.682 in a shake flask containing 100 ml of the following defined medium: casamino acids (1.4 g / L); yeast extract (2 g / L); (NH4)2SO4 (1.85 g / L); K2HPO4 (30 g / L); MgSO4.7H2O (2 g / L); thiamine.HCl (0.015 g / L); glucose (18.1 g / L); trace elements solution (3 ml / L) consisting of FeCl3.6H2O, 27 g; ZnCl2.4H2O, 1.3 g, CoCl2.H2O, 2 g; Na2Mo4.2H2O, 2g; CaCl2.2H2O, 1 g; CuCl2.2H2O, 1 g; H3BO3, 0.5 g; distilled H2O, 1000 ml; and HCl, 100 ml. In addition, 0.0156 g / L of ZnCl was added to trace minerals to make the concentration of Zn five times greater in the shake flask and fermentor. Kanamycin (50 μg / L) was added as an antibiotic. The shake flask culture was used to inoculate a 5-L BioFlo III fermentor (New Brunswick Scientific, Edison, N.J.) containing 4.3 L of the medium described above. Later in the growth (5.5 hr), 14.1 g / L of casamino...

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Abstract

The present invention relates to the construction, expression, and purification of synthetic or recombinant light chain (LC) botulinum neurotoxin genes from all botulinum neurotoxin serotypes. The methods of the invention can provide 1.1 g of the LC per liter of culture. The LC product is stable and proteolytically active. Methods of using the products of the invention are described.

Description

SPECIFICATION[0001]This application is a divisional of U.S. patent application Ser. No. 10 / 011,588 filed Nov. 6, 2001, which is a continuation-in-part of U.S. patent application Ser. No. 09 / 910,186 filed Jul. 20, 2001, which is a continuation of U.S. patent application Ser. No. 09 / 611,419 filed Jul. 6, 2000, which is a continuation of U.S. patent application Ser. No. 08 / 123,975, filed Sep. 21, 1993, wherein said application Ser. No. 09 / 611,419 is based on U.S. Provisional Applications Nos. 60 / 133,866, 60 / 133,868, 60 / 133,869, 60 / 133,865, 60 / 133,873, and 60 / 133,867, all filed May 12, 1999, all of which are incorporated herein by reference in their entirety. The instant application is also based on U.S. Provisional Application No. 60 / 246,774, filed on Nov. 6, 2000, and U.S. Provisional Application No. 60 / 311,966 filed Aug. 9, 2001, which are incorporated herein in their entirety by reference.FIELD OF THE INVENTION[0002]This invention is directed to construction, expression, and purific...

Claims

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Application Information

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IPC IPC(8): C07K14/33C12N9/64
CPCC07K14/33C12N9/6489
Inventor SMITH, LEONARD AJENSEN, MELODY
Owner SMITH LEONARD A